Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

Abstract

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment

“…Specifically, we adapted a comet assay protocol (RICom) for use with reconstructed 3D EpiIntestinal™ microtissues (MatTek Corporation) and demonstrated that this protocol offers a suitable technology platform for identifying ingested genotoxic hazards for human ­intestinal tissues. The RICom assay protocol is informed by the well-established 3D Skin Comet assay published by Pfuhler et al [ 15 ], OECD in vivo comet guideline [ 5 ], and the recently published RICyt micronucleus test [ 31 ]. The 3D EpiIntestinal™ model is a reconstructed mimic of human small intestine tissue that retains native tissue architecture (microvilli), ­polarization (basement membrane, villin), heterogenous cell populations (enterocytes, paneth cells, M cells, tuft cells, intestinal stem cells), barrier function, and biochemical activity akin to native human intestinal tissue [ 16 ].…”

“…With further validation work, the RICom could be used as a bridging model between in vitro 2D and in vivo comet assay for genotoxic hazard identification through the intestinal route of exposure. The RICom assay complements the recently developed Reconstructed Intestinal Micronuclei Cytome (RICyt) assay [ 31 ].…”

“…Cell recovery solution (Corning, Tewksbury, MA) and human tumour dissociation kit (130-095-929, Miltenyi Biotec, Bergisch Gladbach, Germany) were used for the purpose of 3D tissue dissociation as previously described [ 31 ].…”

“…Tissue dissociation was performed as previously described in Lim et al [ 31 ] where intact cells were fixed and examined under a microscope. Transwell™ inserts were removed and washed with pre-warmed HBSS +Ca/Mg to remove cell debris, remaining culture media and test material.…”

“…Accepting this, most in vivo comet testing is performed in rodents [ 11–13 , 30 ]; an animal model increasingly acknowledged to offer lesser physiological relevance for humans. Our particular assay protocol was developed and evaluated as an extension of our recently published micronuclei cytome (RICyt) assay protocol [ 31 ].…”